Upregulation of Hsp72 mediates anoxia/reoxygenation neuroprotection in the freshwater turtle via modulation of ROS.

The neuroprotective role of Hsp72 has been demonstrated in several ischemic/stroke models to occur primarily through mediation of apoptotic pathways, and a number of heat shock proteins are upregulated in animal models capable of extended anoxic survival. In the present study, we investigated the role of Hsp72 on cell death and apoptotic regulators in one anoxia tolerant model system, the freshwater turtle Trachemys scripta. Since Hsp72 is known to regulate apoptosis through interactions with Bcl-2, we manipulated the levels of Hsp72 and Bcl-2 with siRNA in neuronally enriched primary cell cultures and examined downstream effects. The knockdown of either Hsp72 or Bcl-2 induced cell death during anoxia and reoxygenation. Knockdown of Bcl-2 resulted in increases in apoptotic markers and increased ROS levels 2-fold. However, significant knockdown of Hsp72 did not have any effect on the expression of key mitochondrial apoptotic regulators such as Cytochrome c and caspase-3. Hsp72 knockdown however significantly increased apoptosis inducing factor in both anoxia and reoxygenation and resulted in a six-fold induction of hydrogen peroxide levels. These findings suggest that the neuroprotection offered by Hsp72 in the anoxia/reoxygenation tolerant turtle is through the mediation of ROS levels and not through modulation of caspase-dependent pathways.